This invention relates to 1-heteroaryl-pyrrolidine, -piperidine and -homopiperidine derivatives and to processes for the preparation of, intermediates used in the preparation of, compositions containing and the uses of, such derivatives.
It has been reported that the immunosuppressant FK-506 promotes neurite outgrowth in vitro in neuronal cell line and culture models (see Lyons et al, Pro. Natl. Acad. Sci., 1994, 91, 3191-95 and Snyder et al, Nature Medicine, 1995, 1, 32-37). International Patent Applications publication nos. WO 96/40140, WO 96/40633 and WO 97/16190 disclose compounds that have neurotrophic activity but which lack inhibitory action at the protein phosphatase calcineurin and therefore which have no immunosuppressive activity.
It has been suggested in International Patent Applications publication numbers WO 96/40140 and WO 96/40633 that the neurotrophic effect of these compounds is mediated, at least in part, by a high affinity interaction with the FK-506 binding proteins, such as FKBP-12 or FKBP-52. However, the mechanism by which this interaction with FKBP-type immunophilins results in a neurotrophic effect is at present unknown. The range of neurotrophic activity that can be realised through this neurotrophic/non-immunosuppressant class of compounds has been explored and it has been found that axon regeneration can be promoted after facial nerve crush and sciatic nerve crush in the rat. It has also been observed that the functional regeneration of dopamine neurons damaged with the toxin MPTP was promoted by the compounds disclosed therein in mice. Additionally, it was reported that restoration of striatal innervation in the rat was promoted by the compounds disclosed therein following 6-hydroxydopamine lesioning of dopaminergic neurons (see Hamilton and Steiner, Current Pharmaceutical Design, 1997, 3, 405-428).
International Patent Applications publication numbers WO 98/00278, WO 98/13343, WO 98/13355, WO98/20891, WO98/20892 and WO98/20893 describe various neurotrophic pyrrolidine, piperidine and homopiperidine derivatives having an acyl, amide, oxalyl, or similar linking group, at the 1-position of the heterocycle.
U.S. Pat. No. 5,721,256 describes various pyrrolidine, piperidine and homopiperidine derivatives having an SO2 linking group at the 1-position of the heterocycle, as having affinity for rotamase enzymes.
European Patent Application publication number 0 657 451 A2 generically discloses a number of 2-(1-pyrrolidino)-benzoxazoles as leukotriene biosynthesis inhibitors, and specifically discloses methyl 1-(5-chloro-2-benzoxazolyl)proline.
It has now been found that the presently-disclosed substances are neurotrophic agents which have an affinity for FKBP-type immunophilins. In particular, they are potent inhibitors of the enzyme activity and especially of the cis-trans prolyl isomerase (rotamase) activity of FKBP-type immunophilins, particularly the immunophilins FKBP-12 and FKBP-52. The present substances do not significantly inhibit the protein phosphatase calcineurin and therefore lack any significant immunosuppressive activity.
The present substances moderate neuronal degeneration and promote neuronal regeneration and outgrowth and as such can be used for treating neurological disorders arising from neurodegenerative diseases or other disorders involving nerve damage. The neurological disorders that may be treated include senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neurone disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases.
Preferably, the present substances can be used for treating senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neurone disease, Parkinson""s disease, Huntingdon""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus.
The substances of the present invention are compounds of the formula (I): 
or a pharmaceutically acceptable salt, or solvate of either entity, wherein:
X is O, S, NH or N(Cl1-6 alkyl);
R1, R2, R3 and R4 are each independently H, OH, OCO(C1-6 alkyl), CO2(C1-6 alkyl), CONH2, CONH(C1-6 alkyl), CON(C1-6 alkyl)2, halo, C3-7 cycloalkyl, C3-7 cycloalkyloxy, C2-6 alkenyl aryl1, C1-6 alkyl optionally substituted by one or more substituents selected from halo and C3-7 cycloalkyl, and C1-6 alkoxy optionally substituted by one or more substituents selected from fluoro and C3-7 cycloalkyl;
A is unbranched C3-5 alkylene optionally substituted by up to three C1-6 alkyl groups;
D is O or S;
E is O, S, NH, N(C1-6 alkyl) or CR11R12;
G is C1-4 alkyl or C2-14 alkenyl, each of which is optionally substituted by one or more substituents independently selected from halo, aryl, C1-4 alkoxy, cycloalk, het and NR5R6,
R5 and R6 are either each independently H or C1-6 alkyl, or are taken together to form, with the nitrogen atom to which they are attached, a 4 to 7-membered heterocyclic ring optionally containing another hetero-moiety selected from NR7, O and S(O)p, and which 4 to 7-membered heterocyclic ring is optionally substituted by up to 3 substituents independently selected from C1-6 alkyl and C1-6 alkoxy;
R7 is H, C1-6 alkyl, C2-6 alkenyl, COR8, SO2R8, CONR9R10, CO2R8 or SO2NR9R10;
R8 is C3-7 cycloalkyl, C2-6 alkenyl, aryl1, or C1-6 alkyl optionally substituted by C3-7 cycloalkyl or aryl1;
R9 and R10 are each independently H, C2-6 alkenyl, C3-7 cycloalkyl, or C1-6 alkyl optionally substituted by C3-7 cycloalkyl or aryl;
R11 and R12 are each independently H, aryl, C2-8 alkenyl or C1-8 alkyl, wherein said C2-8 alkenyl and C1-8 alkyl groups are optionally substituted by one or more substituents independently selected from halo, NO2, C1-6 alkyl, C2-6 alkenyl, cycloalk, OH, C1-6 alkoxy, C2-6 alkenyloxy, phenyloxy, benzyloxy, NH2, aryl and het;
p is 0, 1 or 2;
wherein xe2x80x9carylxe2x80x9d means phenyl or naphthyl, each of which is optionally substituted by up to 3 substituents independently selected from C1-6 alkyl optionally substituted by one or more halo or C3-7 cycloalkyl groups, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, OH, halo, NO2, phenyloxy, benzyloxy, phenyl and NH2;
xe2x80x9caryl1xe2x80x9d means phenyl, naphthyl or benzyl, each of which is optionally substituted by 1 or 2 substituents independently selected from C1-6 alkyl optionally substituted by one or more halo or C3-7 cycloalkyl groups, C1-6 alkoxy and halo;
xe2x80x9ccycloalkxe2x80x9d is C3-8 cycloalkyl optionally substituted by up to 3 substituents independently selected from C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, OH, halo, and C,6 alkyl optionally substituted by one or more halo;
and xe2x80x9chetxe2x80x9d means a 5- or 6-membered monocyclic, or 8-, 9- or 10-membered bicyclic heterocycle containing 1 to 3 heteroatoms independently selected from O, N and S, which is optionally substituted by up to 3 substituents independently selected from C1-6 alkyl optionally substituted by one or more halo or C3-7 cycloalkyl groups, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, OH, halo, NO2, phenyloxy, benzyloxy and NH2;
with the proviso that the compound is not methyl 1-(5-chloro-2-benzoxazolyl)proline.
Throughout the above definitions, xe2x80x9chaloxe2x80x9d means fluoro, chloro, bromo or iodo. Alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, except where indicated, can be unbranched- or branched-chain, where the number of carbon atoms allows.
It is to be appreciated herein that where X is NH, in certain conditions the NH proton can be mobile and can reside on the other nitrogen in the benzimidazole ring, viz. formula (IA) below: 
It is to be understood that all such compounds of formula (IA) are included in the scope of the compounds of formula (I) as tautomers thereof.
The pharmaceutically acceptable salts of the compounds of the formula (I) include the acid addition and the base salts thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and examples are the sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine salts.
For a review on suitable salts see for example Berge et al, J. Pharm. Sci., 1977, 66, 1-19.
The pharmaceutically acceptable solvates of the compounds of the formula (I) include the hydrates thereof.
Also included within the present scope of the substances of the invention are polymorphs and radiolabelled derivatives thereof.
A compound of the formula (I) contains one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. The present invention includes the individual stereoisomers of the compounds of the formula (I) and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Certain of the compounds of formula (I) can exist as geometric isomers. The present invention includes the individual geometric isomers of the compounds of the formula (I), together with mixtures thereof.
Separation of diastereoisomers and geometric isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a mixture of isomers of a compound of the formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of the formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
Certain of the compounds of formula (I) can exist as tautomers. It is to be understood that the invention encompasses all individual tautomers of the compounds of formula (I), together with mixtures thereof.
Preferably X is O or NH.
Preferably at least two of R1, R2, R3 and R4 are H.
Preferably R1 is H, halo or CO2(C1-6 alkyl).
More preferably R1 is H or CO2CH3.
Preferably R2 is H, halo, C3-7 cycloalkyl, C3-7 cycloalkyloxy, C2-6 alkenyl, C1-6 alkyl optionally substituted by one or more substituents selected from halo and C3-7 cycloalkyl, or C1-6 alkoxy optionally substituted by one or more substituents selected from fluoro and C3-7 cycloalkyl.
More preferably R2 is H, halo, C1-6 alkyl optionally substituted by one or more substituents selected from halo and C3-7 cycloalkyl, or C1-6 alkoxy optionally substituted by one or more C3-7 cycloalkyl groups.
Yet more preferably R2 is H, halo, C1-4 alkyl optionally substituted by one or more halo, or C1-4 alkoxy.
Even more preferably R2 is H, F, I, Br, Cl, CH3, C2H5, CH2CH(CH3)2, CF3, OCH3or OCH(CH3)2.
Most preferably R2 is H, F, Cl, Br, I or CF3.
Preferably R3 is H, halo, C3-7 cycloalkyl, C3-7 cycloalkyloxy, C2-6 alkenyl, C1-6 alkyl optionally substituted by one or more substituents selected from halo and C3-7 cycloalkyl, or C1-6 alkoxy optionally substituted by one or more substituents selected from fluoro and C3-7 cycloalkyl.
More preferably R3 is H, halo, C1-6 alkyl optionally substituted by one or more substituents selected from halo and C3-7 cycloalkyl, or C1-6 alkoxy optionally substituted by one or more C3-7 cycloalkyl groups.
Yet more preferably R3 is H, halo, C1-4 alkyl optionally substituted by one or more halo, or C1-4 alkoxy.
Even more preferably R3 is H, F, I, Br, Cl, CH3, C2H5, CH2CH(CH3)2, CF3, OCH3or OCH(CH3)2.
Most preferably R3 is H, F, Cl, Br, I or CF3.
When X is O or NH, R2 and R3 are preferably each independently H, halo or CF3.
Preferably R4 is H, halo or C1-6 alkyl.
More preferably R4 is H or CH3.
Preferably A is unbranched C3-5 alkylene optionally substituted by a C1-6 alkyl group.
More preferably A is unbranched C3-5 alkylene.
Most preferably A is butylene, i.e. (CH2)4.
Preferably D is O.
Preferably E is NH or N(C1-6 alkyl).
Most preferably E is NH.
Preferably G is C1-4 alkyl or C2-14 alkenyl, each of which is mono- or disubstituted by substituents independently selected from het, aryl, cycloalk or NR5R6.
More preferably G is C2-4 alkyl or C2-4 alkenyl, each of which is terminally monosubstituted by NR5R6.
Yet more preferably G is C2-4 alkyl or C2-4 alkenyl, each of which is terminally substituted by NR5R6, where R5 and R6 are either each independently H or C1-6 alkyl, or are taken together to form, with the nitrogen atom to which they are attached, a 5 to 7-membered ring optionally containing another hetero-moiety selected from NR7 or O, and which ring is optionally substituted by up to 3 substituents independently selected from C1-6 alkyl and C1-6 alkoxy, and wherein R7 is H, C1-6 alkyl, COR8 or CONR9R10.
Even more preferably G is (CH2)mNR5R6, where m is 2, 3 or 4, and R5 and R6 are either each both H, or are taken together to form, with the nitrogen atom to which they are attached, a 6-membered ring optionally containing another hetero-moiety at the 4-position relative to the ring nitrogen directly attached to the (CH2)m moiety, which hetero-moiety is selected from NH, NCOCH3, NCH3, NCONHCH(CH3)2 or O, and which ring is optionally substituted by up to 2 CH3 substituents on the ring atoms adjacent to the ring nitrogen directly attached to the (CH2)m moiety.
Further more preferably, G is (CH2)2NR5R6, where R5 and R6 are both H or are taken together taken together to form, with the nitrogen atom to which they are attached, a 6-membered ring optionally containing another hetero-moiety at the 4-position relative to the ring nitrogen directly attached to the (CH2)m moiety, which hetero-moiety is selected from NH, NCOCH3, NCH3, NCONHCH(CH3)2 or O, and which ring is optionally substituted by up to 2 CH3 substituents on the ring atoms adjacent to the ring nitrogen directly attached to the (CH2)2 moiety.
Most preferably G is (CH2)2NR5R6, where NR5R6 is piperidino, morpholino, piperazino, 
where R13 is H, COCH3, CH3, or CONHCH(CH3)2.
Preferably the compounds have the stereochemistry shown in formula (IB) below. 
A preferred group of substances are those where the substituents X, A, D, E, G, R1, R2, R3 and R4 have the values found in the Examples below, and the stereochemistry is as shown above in formula (IB).
The most preferred group of substances are the compounds of the Examples below and their salts and solvates.
Particularly preferred substances are the compounds of Examples 3, 10, 11, 12 16, 17, 18, 19, 20, 22, 27 and 31, and the salts and solvates thereof.
The compounds of the formula (I) can be prepared by a number of methods using conventional procedures such as by the following illustrative methods, and suitable adaptation thereof. Such methods are a further aspect of the invention.
Unless otherwise specified below, the substituents are as defined for the compounds of formula (I) above.
Method 1
All the compounds of formula (I) can be made via reaction of a compound of the formula (II) (including regioisomers thereof (IIA) where appropriate) below where X1 is O, S, N(C1-6 alkyl) or N(APG), where xe2x80x9cAPGxe2x80x9d is an amino-protecting group which can be readily removed to give the corresponding NH compound, and L1 is a suitable leaving moiety such as Cl, Br, I, SH, SCH3, SO2CH3, SO2CF3, OSO2CH3 or OSO2CF3, with a compound of formula (III) below. 
Suitable amino-protecting groups are well-known to the skilled chemist and are exemplified in xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d by T W Greene and P G M Wuts, John Wiley and Sons Inc., 1991, herein incorporated by reference. Preferably the amino-protecting group is the t-butyloxycarbonyl (xe2x80x9cBocxe2x80x9d) group, which can be readily removed either in situ during the course of the reaction between (II) and (III) above, or by later treatment with trifluoroacetic acid (TFA), in a suitable solvent such as dichloromethane.
Typically the reaction is carried out by heating the subtrates (II) (including regioisomers (IIA) thereof where appropriate) and (III) together in a suitable organic solvent such as dimethylacetamide, to a temperature in the range 25-200xc2x0 C., preferably around 80xc2x0 C., optionally in the presence of a base such as triethylamine or diisopropylethylamine, also optionally in the presence of a metal such as copper.
Compounds of formulae (II) (including regioisomers (IIA) thereof where appropriate) and (III) are available by conventional methods such as those exemplified in the Preparations below.
Method 2
Compounds of the formula (I) where X is O or S, D is O and E is O, S, NH or N(C1-6 alkyl), can be prepared by reaction of a compound of the formula (IV): 
where X is O or S, and L2 is a suitable leaving group such as azide, mesylate, tosylate, OH, Cl, Br, I, etc., including where the COL2 moiety is a suitable activated ester, with a compound of formula Gxe2x80x94Exe2x80x94H, or salt thereof. Examples of such activated esters can be derived from the parent acid (IV; L2 is OH), for example by reaction with a hydroxybenzotriazole-type reagent such as 1-hydroxybenzotriazole, and a carbodiimide reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. These types of acid-activating reagents can be used alone or in combination. Examples of hydroxybenzotriazole based reagents which can be used by themselves are benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate and O-(1H-benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate. Further examples of L2 include the moieties derived from reaction of (IV; L2 is OH) with pentafluorophenol and N-hydroxysuccinimide. Similarly, L2 moieties can be used which make compounds of formula (IV) a mixed anhydride and examples include the compounds derived from reaction of compounds of formula (IV; L2 is OH) with reagents such as isobutylchloroformate and bis(2-oxo-3-oxazolidinyl)phosphinic chloride. Additionally, L2 can be imidazolyl, such compounds being derived from reaction of compounds of formula (IV; L2 is OH), with N,Nxe2x80x2-carbonyidiimidazole.
The reaction of compounds of formula (IV) with compounds of formula Gxe2x80x94Exe2x80x94H is suitably carried out in a suitable solvent in the presence of an optional base, such as N-methylmorpholine
Additionally, compounds of the formula (I), where X is O or S, D is O and E is O, NH or N(C1-6 alkyl), may be prepared by directly heating together compounds of the formula (IV) (including tautomers thereof where appropriate), for example where L2 is OH, with compounds of the formula Gxe2x80x94Exe2x80x94H, where E is O, NH or N(C1-6 alkyl), optionally in the presence of a catalyst, such as a suitable acid or base, and optionally in an appropriate solvent.
Additionally, in an analogous synthesis, compounds of formula (I) where X is NH can be made via reaction of a compound of formula (IVA) or (IVB): 
where X2 is N-APG, where APG is defined as for Method 1 above.
Compounds of formulae (IVA) and (IVB) where X2 is N-Boc and L2 is OH can in certain circumstances, for example in the presence of certain dehydrating systems such as in a hydroxybenzotriazole/1-(3-dimethylamino)-3-ethylcarbodiimide hydrochloride/N-methylmorpholine in a suitable organic solvent such as dichloromethane, form compounds of fomulae (IVC) and (IVD): 
which may be stable and isolatable.
Compounds of formula (IV) (including tautomers thereof where appropriate) may be prepared by standard methods, such as that outlined in the Preparations below, and suitable variation thereof.
Compounds of formula Gxe2x80x94Exe2x80x94H are commercially available or are otherwise available via conventional routes, such as are described in the Preparations below.
Method 3
Compounds of the formula (I) where E is CR11R12 can be prepared by reaction of a compound of the formula (IV) (including tautomers thereof where appropriate) as defined above, with an organometallic species MNCR11R12G, where n is 1 or less, depending on the valence of the metallic species M. M can be a single metal or a combination of metals, optionally with other ligands such as halides (e.g. Grignard-type reagents). An example of this type of reaction is where L2 is a halide, M is CuLi and n is 0.5. This type of reaction is described in xe2x80x9cAdvanced Organic Chemistryxe2x80x9d by J. March, 3rd edition, Wiley Interscience in sections 0-106 and 0-107 and the references therein, herein incorporated by reference.
Method 4
Compounds of formula (I) where D is S (including tautomers thereof where appropriate) can be made from the corresponding compound of formula (I) where D is O (including tautomers thereof where appropriate) by reaction with a sulphur nucleophile such as those mentioned in xe2x80x9cAdvanced Organic Chemistryxe2x80x9d by J March, Wiley-Interscience, 1985, section 6-11, and the references therein, herein incorporated by reference.
A suitable reagent for carrying out such a transformation is 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulphide (Lawesson""s reagent). For a review of this reagent and reaction, see for instance Pederson, et al, Bull. Chim. Soc. Belges 87, 223 (1978).
Method 5
Compounds of the formula (I) are available via reaction of compounds of formula (V) and (VI): 
wherein L3 is a suitable leaving group such as Cl, Br, or I. The reaction is suitably carried out in the presence of an additional base such as triethylamine, and in a suitable aprotic organic solvent such as dichloromethane.
The reaction in some circumstances, i.e. specific substituents, solvents, bases, reaction conditions, etc., will proceed directly to give the compound of formula (I). In other circumstances the formation may proceed in a stepwise manner via intermediates of formulae (VIl) or (VIII), or salts thereof: 
which may be stable and isolatable.
Compounds of formulae (V) and (VI) are available commercially or via standard methods known in the art, or suitable adaptation thereof.
Certain of the subtances of the invention may be interconverted into other substances of the invention by conventional functional group interconversion methods.
It will be appreciated that all the substances of the invention are available via methods known in the art and the methods outlined and exemplified herein and suitable adaptation thereof using methods known in the art. The skilled chemist will exercise his skill and judgement as to any necessary adaptation, for instance in the choice of reagents, conditions, compatability of substrates and reagents with desired reaction, order of reaction steps, protection/deprotection, further reactions, etc.
It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during the synthesis of substances of the invention. These steps may be acheived by conventional techniques, for example as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by T W Greene and P G M Wuts, John Wiley and Sons Inc., 1991.
Some of the reaction steps outlined herein could result in racemisation at certain sensitive stereochemical centres, if present. The compound with the desired stereochemistry may be made for example by subsequent resolution using conventional methods such as by chiral HPLC, or by instead carrying out the relevant transformation in a manner which does not lead to racemisation, for example by use of a chiral auxiliary in the reactant.
All of the above reactions and the preparations of novel starting materials used in the preceding methods are conventional and appropriate reagents and reaction conditions for their performance or preparation as well as procedures for isolating the desired products will be well-known to those skilled in the art with reference to literature precedents and the Examples and Preparations herein.
A pharmaceutically acceptable salt of a compound of the formula (I) may be readily prepared by mixing together solutions of a compound of the formula (I) and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
The affinity of the compounds of the formula (I) for FKBP-12 can be determined in vitro in a coupled colorimetric PPlase assay using similar procedures to published methods (e.g. see Kofron, J. L., et al., Biochemistry, 1991, 30, 6127-6134, Zarnt, T., et al., Biochem. J. 1995, 305, 159-164, Holt, D. A., et al., J. Am. Chem. Soc., 1993, 115, 9925-9938). In these methods, the cis-trans isomerisation of a hydrophobic amino acid-proline bond in a tetrapeptide substrate (e.g. the phenylalanine-proline bond in N-succinyl-ala-phe-pro-phe-p-nitroanilide [succinyl-AFPF-pNA]) can be determined by monitoring cleavage of pNA from the transPro-containing peptide by an excess of chymotrypsin.
The IC50 (the concentration of the compound of the formula (I) producing 50% inhibition) values were determined using the following assay methodology. Assay buffer (2.175 ml) (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), 100 mM NaCl, 1 mM dithiothreitol (DTT), pH 8.0) is equilibrated to 10xc2x0 C. in a cuvette. 12.5 xcexcl of a solution of the present compound in DMSO, 250 xcexcl of a 60 mg/ml solution of xcex1-chymotrypsin in 1 mM aqueous hydrochloric acid and then 50 xcexcl of a solution of human recombinant FKBP-12 (4.5 xcexcM) in assay buffer are added and mixed. The reaction is initiated by addition of 12.5 xcexcl of a solution of 20 mM succinyl-AFPF-pNA in DMSO. The absorbance at 390 nM is monitored for one minute collecting data every 0.25 second. Data are fitted with a first order rate equation with offset and the rate constant obtained corrected for the rate of uncatalysed isomerisation of the substrate. The rate constant determined at different inhibitor concentrations (10 nM to 100 xcexcM) is expressed as % inhibition of the control rate constant. The IC50 is estimated using a non-linear least squares curve fitting routine of the sigmoidal dose response data.
Ki,app (the apparent inhibition constant) was determined for the present compounds using the assay procedure described below. Assay buffer (2.175 ml) (50mM HEPES, 100 mM NaCl, 1 mM DTT, pH 8.0) is equilibrated to 10xc2x0 C. in a cuvette. 12.5 xcexcl of a solution of the present compound in DMSO, 250 xcexcl of a 60 mg/ml solution of xcex1-chymotrypsin in 1 mM aqueous hydrochloric acid and then 50 xcexcL of a solution of human recombinant FKBP-12 (1.5 xcexcM) in assay buffer are added and mixed. The reaction is initiated by adding 12.5 xcexcl of a solution of anhydrous succinyl-AFPF-pNA (100 xcexcM final concentration) in a 400mM solution of LiCl in trifluoroethanol. The absorbance at 390 nM is monitored for 3 minutes collecting data every 0.5 second. Data are fitted with a first order rate equation with offset and the initial velocity (v) is calculated from the concentration of cis (re leu-pro bond)-succinyl-AFPF-pNA at to and the first order rate constant at different inhibitor concentrations (I). Data in the form Vinh/Vcontrol v. [I] are fitted with an equation for reversible tight binding inhibition to generate values for Ki,app (see Morrison, J. F., et al, Comments Mol. Cell Biophys., 1985, 2, 347-368). This analysis is used when the Ki,app approaches the concentration of FKBP-12 in the assay (30 nM). Dixon analysis (see Dixon, M., Biochem. J.,1953, 55, 170-171) is used for generating values of Ki,app for less potent compounds.
The same methodology is used to measure Ki,app for FKBP-52, with the following modifications: 40 xcexcl human recombinant FKBP-52 (5.2 xcexcM) is substituted for FKBP-12 and 2.185 ml assay buffer are used in the assay.
The neurite outgrowth promoting activity of the compounds of the formula (I), without proviso, can be determined in explant cultures of embryonic chick dorsal root ganglia. Dorsal root ganglia (DRG) are isolated aseptically according to the method of Bray (see xe2x80x9cCulturing Nerve Cellsxe2x80x9d, Ed. G. Banker and K. Goslin, MIT Press, Cambridge, Mass., 1991, p.119). The individual ganglia were kept in Ca2+/Mg2+-free Tyrodes buffer on ice until a number of ganglia had been collected. Individual ganglia were then transferred into collagen-coated 24-well culture plates containing Neurobasal medium plus B27 supplements and incubated at 37xc2x0 C. in a 5% CO2 atmosphere. The test substance was added after allowing 4 hours for the ganglia to attach. The explants were fixed and stained with Coomassie blue after 24 or 48 hours in culture. For each treatment 4 to 6 ganglia were analysed and scored by estimating the extent of neurite outgrowth relative to the diameter of the explant using image analysis. The present substances were tested with and without 10 ng/ml nerve growth factor (NGF) present and compared to outgrowth in the presence of 10 ng/ml nerve growth factor alone.
An alternative system for measuring neurite outgrowth promoting activity of FKBP-12 PPlase inhibitors is the SH-SY-5Y neuroblastoma model described by Gold, B. G., et al, in Exp. Neurol., 1997, 147(2), 269-278. Cells are maintained in Dulbecco""s Modified Eagle""s Medium (DMEM) supplemented with 10% foetal calf serum (FCS), 50 U/ml penicillin, 50 xcexcg/ml streptomycin at 37xc2x0 C. in a 7% CO2 atmosphere. Cells are plated at 1xc3x97106 cells per well and treated for 5 days with 400 nM aphidicolin. Cells are then washed and treated with NGF at 10 ng/mlxc2x1various compound concentrations for 7 days to determine if the compounds promote neurite outgrowth in the presence of suboptimal NGF concentrations (and/or in the absence of NGF). Neurite outgrowth is determined by using image analysis to measure neurite lengths in 20 random fields.
The neurotrophic activity of the present substances can be evaluated in vivo using the sciatic nerve crush model in rat as a model for peripheral nerve regeneration (see Bridge, P. M., et al., Experimental Neurology, 1994, 127, 284-290, Medinaceli, L., et al., Expl. Neurology, 1982, 77, 634-643, Gold, B. G., et al., Restorative Neurology and Neuroscience, 1994, 6, 287-296), the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine models in various species as a model for regeneration in Parkinson""s disease (see Mokry, J., Physiol. Res., 1995, 44(3), 143-150) and fimbria-fornix lesions as a model for regeneration in Alzheimer""s disease (see Cassel, J. C., Duconseille, E., Jeltsch, H. and Will, B., Prog. Neurol., 1997, 51, 663-716).
The substances of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
For example, the substances of the invention can be administered orally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate or controlled release applications.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose or milk sugar as well as high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the substances of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The substances of the invention can also be injected parenterally, for example, intravenously, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
For oral and parenteral administration to human patients, the daily dosage level of the substances of the invention will usually be from 1 xcexcg/kg to 25 mg/kg (in single or divided doses).
Thus tablets or capsules of the may contain from 0.05 mg to 1.0 g of active substance for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
The substances of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container or a nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A [trade mark] or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA [trade mark]), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a substance of the invention and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each metered dose or xe2x80x9cpuffxe2x80x9d contains from 20 xcexcg to 20 mg of a substance of the invention for delivery to the patient. The overall daily dose with an aerosol will be in the range of from 20 xcexcg to 20 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
Alternatively, the substances of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The substances of the invention may also be transdermally administered by the use of a skin patch. They may also be administered by the ocular route, particularly for treating neurological disorders of the eye.
For ophthalmic use, the substances can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
For application topically to the skin, the substances of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyidodecanol, benzyl alcohol and water.
The substances of the invention, without proviso, can also be administered together with other neurotrophic agents such as neurotrophic growth factor (NGF), glial derived growth factor, brain derived growth factor, ciliary neurotrophic factor and/or neurotrophin-3. The dosage level of the neurotrophic agent will depend upon the neurotrophic effectiveness of the combination and the route of administration used.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
Thus the invention further provides:
(i) a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt or solvate thereof, together with a pharmaceutically acceptable excipient, diluent or carrier, optionally also containing another neurotrophic agent;
(ii) a compound of the formula (I) or a pharmaceutically acceptable salt, solvate or composition thereof, for use as a medicament;
(iii) the use of a compound of the formula (I), without proviso, or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the treatment of neuronal degeneration;
(iv) the use of a compound of the formula (I), without proviso, or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the promotion of neuronal regeneration and outgrowth;
(v) the use of a compound of the formula (I), without proviso, or of a pharmaceutically acceptable salt, solvate or composition thereof, for the manufacture of a medicament for the treatment of a neurological disease or disorder such as a neurodegenerative disease;
(vi) use as in (v) where the neurological disease or disorder is selected from the group consisting of senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neuron disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases;
(vii) use as (vi) where the neurological disease or disorder is senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neuron disease, Parkinson""s disease, Huntington""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus;
(viii) a method of treatment of a human to treat neuronal degeneration which comprises treating said human with an effective amount of a compound of the formula (I), without proviso, or with a pharmaceutically acceptable salt, solvate or composition thereof;
(ix) a method of treatment of a human to promote neuronal regeneration and outgrowth which comprises treating said human with an effective amount of a compound of the formula (I), without proviso, or with a pharmaceutically acceptable salt, solvate or composition thereof;
(x) a method of treatment of a human to treat a neurological disease or disorder such as a neurodegenerative disease which comprises treating said human with an effective amount of a compound of the formula (I), without proviso, or with a pharmaceutically acceptable salt, solvate or composition thereof;
(xi) a method as in (x) where the neurological disease or disorder is selected from the group consisting of senile dementia (Alzheimer""s disease) and other dementias, amyotrophic lateral sclerosis and other forms of motor neuron disease, Parkinson""s disease, Huntington""s disease, neurological deficits associated with stroke, all forms of degenerative disease affecting the central or peripheral nervous system (e.g. cerebellar-brainstem atrophies, syndromes of progressive ataxias), all forms of muscular dystrophy, progressive muscular atrophies, progressive bulbar muscular atrophy, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), herniated, ruptured or prolapsed intervertebrae disc syndromes, cervical spondylosis, plexus disorders, thoracic outlet syndromes, all forms of peripheral neuropathy (both diabetic and non-diabetic), trigeminal neuralgia, glossopharyngeal neuralgia, Bell""s Palsy, all forms of auto-immune related disease resulting in damage of the central or peripheral nervous system (e.g. multiple sclerosis, myasthenia gravis, Guillain-Barrxc3xa9 syndrome), AIDS related disorders of the nervous system, dapsone ticks, bulbar and retrobulbar affections of the optic nerve (e.g. retinopathies and retrobulbar neuritis), hearing disorders such as tinnitus, and prion diseases; and
(xii) a method as in (xi) where the neurological disease or disorder is senile dementia (Alzheimer""s disease) or another dementia, amyotrophic lateral sclerosis or another form of motor neuron disease, Parkinson""s disease, Huntington""s disease, a neurological deficit associated with stroke, physical or traumatic damage to the central or peripheral nervous system (e.g. spinal cord), a peripheral neuropathy (either diabetic or non-diabetic), multiple sclerosis or a hearing disorder such as tinnitus.
The following Examples illustrate the preparation of the compounds of the formula (I). It is to be appreciated that where the compound of the Example and/or Preparation is a benzimidazole, the valence tautomer is also disclosed. In the following Examples and Preparations, room temperature means 20 to 25xc2x0 C. Flash chromatography refers to column chromatography on silica gel (Kieselgel 60, 230-400 mesh). Melting points are uncorrected. 1H Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AC300, a Varian Unity Inova-300 or a Varian Unity Inova-400 spectrometer and were in all cases consistent with the proposed structures. Characteristic chemical shifts are given in parts-per-million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Mass spectra were recorded using a Finnigan Mat. TSQ 7000 or a Fisons Intruments Trio 1000 mass spectrometer. MS means low resolution mass spectrum and the calculated and observed ions quoted refer to the isotopic composition of lowest mass. Hexane refers to a mixture of hexanes (hpic grade) b.p. 65-70xc2x0 C. Ether refers to diethyl ether. Acetic acid refers to glacial acetic acid. Optical rotations were determined at 25xc2x0 C. The nomenclature of the compounds mentioned below was generated by an IUPAC nomenclature program.